Biological nitrogen fixation in the root nodules of leguminous plants is a major component of world food production and therefore practical applications of this field are of major interest.
Prokaryotes can use a wide variety of nitrogen compounds as sole sources of cellular nitrogen. This variety includes ammonia, dinitrogen and nitrate among the inorganic compounds, and proline, arginine and glutamine among complex organic compounds. Each species can utilize a different array of nitrogen compounds. Glutamine, glutamate and aspartate are the key nitrogen compounds in intermediary metabolism. The latter two are the starting compounds of many pathways of amino acid biosynthesis and serve as amino group donors in many reactions. In all other cases the amino group is donated by glutamine. The major enzyme required for the assimilation of ammonia is glutamine synthetase, which catalyses the reaction: EQU Glutamate+NH.sub.3 +ATP .fwdarw.glutamine+ADP+Pi.
Utilization of the assimilated ammonia depends on the activity of glutamate synthase catalyzing: EQU Glutamine+2-ketoglutarate+NADPH .fwdarw.2 glutamate+NADP.sup.+
Since ATP is hydrolysed, these reactions have a favorable equilibrium and allow the use of ammonia in the medium or ammonia derived enzymatically from other nitrogen sources (Meers, J., Tempest, D. and C. Brown (1970) J. Gen. Microbiol. 64:187-194). The formation of ammonia is thus a key step in the biological nitrogen cycle.
Biological nitrogen fixation can be achieved by a variety of microorganisms and occurs through the induction of an enzyme complex, nitrogenase, which converts atmospheric nitrogen to ammonia. This conversion occurs in a group of physiologically diverse prokaryotes, including facultative anaerobes (e.g., Klebsiella pneumoniae) obligate aerobes (e.g., Azotobacter vinelandii), photosynthetic bacteria (e.g., Rhodospirillum rubrum), and some strains of blue-green algae (e.g., Anabaena cylindrica). (Sprent, J. I. (1979) The biology of nitrogen fixing organisms, London, McGraw-Hill, pp. 8-11). While this enzyme complex is common to all characterized nitrogen fixing organisms, the conditions under which it is expressed vary considerably between species (Burns, R. C., Hardy, R. W. F. (1975): Nitrogen fixation in bacteria and higher plants, Springer-Verlag, Berlin).
The first stages of nitrogen fixation, conversion of nitrogen into ammonia, are achieved symbiotically in the root nodules of leguminous plants which contain the nitrogen-fixing bacteria of the genera Rhizobium and Bradyrhizobium (hereinafter referred to as "rhizobial species"). Some non-leguminous plants, e.g., alder, also have interactions with symbiotic bacteria which are nitrogen fixers. In addition, free-living bacteria, e.g., Klebsiella pneumoniae and the photosynthetic blue-green bacteria, also fix nitrogen.
The symbiotic association between plants and bacteria of rhizobial species is the result of a complex interaction between the bacterium and its host, requiring the expression of both bacterial and plant genes in a tightly coordinated manner (Vincent, J. M. (1980) In Symbiotic Associations and Cyanobacteria, Nitrogen Fixation, Vol. 2 (W. E. Newton, W. H. Orme-Johnson, eds.) Baltimore, University Park Press pp. 103-129; and Verma, D. P. S., Legocki, R. P. and S. Auger (1981) In Current Perspectives in Nitrogen Fixation (A. H. Gibson, W. E. Newton, eds.) Canberra: Australian Academy of Science, pp. 205-208). In free-living rhizobial species, nitrogenase synthesis is repressed and is only induced after the symbiotic relationship has been established. Furthermore, some rhizobial species only interact with a narrow range of plant species, whereas other species interact with a wide range.
Bacteria bind to the emerging plant root hairs and invade the root tissue through the formation of an infection thread. The plant responds to this infection by the development of a highly differentiated root nodule. These nodules are the site of synthesis of the nitrogenase complex. Following nitrogen fixation, the fixed nitrogen is exported into the plant tissue and assimilated by the plant-derived enzymes (Scott, D. B., Farnden, K. J. F. and Robertson, J. G. (1976) Nature 263:703-705).
Most rhizobial symbioses are confined to leguminous plants. Furthermore, rhizobial strains which fix nitrogen in association with the agriculturally-important temperate legumes are usually restricted in their host range to a single legume genus. However, some rhizobial strains have been isolated which can fix nitrogen in a diverse group of legume species but can also form an effective symbiosis with non-legumes.
Early literature references to Rhizobium japonicum refer to strains characterized as "slow-growing" Rhizobia. More recent studies of biochemical and genetic characteristics have led to reclassification of "slow-growing" Rhizobia in the genus Bradyrhizobium (Jordan, D. C. (1982) Int. J. Syst. Bacteriol. 32:136). Furthermore, certain "fast-growing" strains have been found which are classified as R. japonicum on the basis of their ability to nodulate Glycine Max cv. Peking, an undeveloped Asian cultivar of soybean. Since the literature sometimes refers to slow-growing (Bradyrhizobium) strains simply as R. japonicum, confusion may occur. For clarity herein, "slow-growing" soybean nodulating strains are termed Bradyrhizobium japonicum strains, while "fast-growing" strains are termed Rhizobium japonicum strains. Similarly, Parasponia Rhizobium sp. has been reclassified as Bradyrhizobium sp. (Parasponia) (see, e.g. Scott, K. F. (1986), "Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia)," Nucl. Acids Res. 14:2905-2919), and will be so referred to herein, although prior art references may specify the former name.
In some cases, e.g., Klebsiella pneumoniae. free-living bacteria can carry out the reduction of nitrogen to ammonia, but in other cases, e.g., Rhizobium trifolii, the reduction occurs when the bacteria are in association with plant roots. Many of these bacterial-plant symbioses are between rhizobial species and legumes and the association is specific. A particular legume species may be nodulated by some rhizobial isolates but not by others. For example, some rhizobial isolates can infect and nodulate soybeans but cannot nodulate garden peas or white clover. The basis of this specificity may involve recognition and binding of the bacterial cell by some component, possibly a lectin, in the plant roots. Thus, fluorescein-labelled soybean binds to 22/25 strains of Bradyrhizobium japonicum which infect soybeans but does not bind to any of the 23 strains from 5 rhizobial species that infect other legumes (Bohlool, B. B. and Schmidt, E. L. (1974) Science 185:269-271).
In addition to recognition specificity, there appear to be a variety of specific host-symbiont interactions which occur during nodulation. For example, rhizobial responses effected by the host plant include morphological changes during conversion to the bacteroid state, and the induction of nitrogenase. Plant responses affected by interaction with rhizobial species include nodule development and synthesis of leghemoglobin. Studies of the temporal unfolding of these responses during nodulation suggest that each stage of the process is mediated by a complex series of feedback signals from the host plant to the bacterial symbiont, and from the bacteria to the plant. Many of these signals appear to be specific for each host-symbiont species pair and account for much of the observed host species specificity of most rhizobial strains.
Despite the ability of certain plants to induce nitrogenase activity in a symbiotic relationship with some rhizobial species the genetic analysis of biological nitrogen fixation has previously been confined to free living nitrogen fixing organisms, in particular Klebsiella pneumoniae. There are 17 linked nitrogen fixation (nif) genes arranged in at least 7 transcriptional units in the nif cluster of Klebsiella (Kennedy, C. et al. (1981) In Current Perspectives in Nitrogen Fixation. (A. H. Gibson, W. E. Newton, eds.) Canberra: Australian Academy of Science, pp. 146-156; and Reidel, G. E. et al. (1979), Proc. Nat. Acad. Sci. U.S.A. 76:2866-2870). Specific designations are assigned to nif genes, e.g. nifH, based on structural homologies to previously identified genes in other nitrogen fixing organisms at the DNA and protein levels. Three of these genes, nifH, nifD and nifK encode the structural proteins of the nitrogenase enzyme complex (viz. the Fe-protein subunit and the .alpha.- and .beta.-subunits of the Mo-Fe protein respectively). These genes are linked on the same operon in K. pneumoniae and are transcribed from a promoter adjacent to the nifH gene. The remainder of the nitrogen fixation genes contain information required for bacterial attachment, root hair curling, initiation and development of nodules and establishment of symbiotic relationships. In addition, regulatory sequences such as promoters, operators, attenuators, and ribosome binding sites are found adjacent to the coding regions. These regulatory sequences control the expression of the structural genes, i.e., the coding sequences downstream in the 3'-direction of the DNA reading strand.
Rhizobium trifolii is an example of a fast-growing rhizobial species with a narrow host range that cannot normally be induced to fix nitrogen in culture. In contrast, a Bradyrhizobium sp. (Parasponia) species has been isolated and this species is a slow-growing organism with a very broad host range capable of an effective symbiotic relationship with a broad variety of tropical legumes as well as the non-legume Parasponia (Ulmaceae) (Trinick, M. J. (1980) J. Appl. Bacteriol. 49:39-53). Bradyrhizobium sp. (Parasponia) can be induced to fix nitrogen in culture although the level of this fixation is about 100-fold less than can be obtained from the free-living Klebsiella pneumoniae. Other slow-growing rhizobial species include the commercially significant Bradyrhizobium japonicum, which nodulates soybeans.
The genetics of biological nitrogen fixation have been well characterized in the free-living organism Klebsiella pneumoniae. The structural genes for nitrogenase (nifH, nifD and nifK encoding the Fe-protein subunit and the .alpha. and .beta. subunits of the Mo-Fe protein, respectively) have been mapped both genetically and physically (Kennedy, C. et al. (1981) supra. and Reidel, G. E. et al. (1979) Proc. Nat. Acad. Sci. U.S.A. 76:2866-2870). Cloned DNA fragments carrying these sequences have been shown, by Southern blot analysis, to hybridize to homologous sequences in a wide range of nitrogen fixing organisms, including rhizobial organisms (Ruvkun, G. B. and F. M. Ausubel (1980) Proc. Nat. Acad. Sci. U.S.A. 77:191-195).
In spite of the ecological diversity of nitrogen fixing organisms, the physiological structure of the nitrogenase enzyme complex appears to be very conserved. In all cases where the enzyme complex has been purified, two proteins are present. The larger protein (dinitrogenase) contains molybdenum, iron and acid-labile sulfur, and carries the binding site for nitrogen and contains two subunit proteins .alpha.- and .beta.-coded by the nifD and nifK genes respectively. The smaller protein (dinitrogenase reductase) contains iron and acid-labile sulfur, and is required for the reduction of the dinitrogenase and for the binding of MgATP used in this reduction. The dinitrogenase reductase is coded by the nifH gene. Chemical and spectral analyses of the purified protein components support a conservation of protein structure between organisms (Scott, K. F. et al. (1981) J. Mol. Appl. Genet. 1:71-81). In some cases the structures are sufficiently similar to allow formation of active hybrid enzymes between purified components, e.g., Azotobacter vinelandii and Klebsiella pneumoniae (Eady, R. R. and B. E. Smith (1979) In: A Treatise on Dinitrogen fixation I, II, eds. Hardy, R. W., Bottomley, F. and R. C. Burns, New York, Wiley Press pp. 399-490). Not surprisingly, therefore, the region of the nif operon coding for nifH and nifD shows homology at the nucleic acid sequence level with the corresponding sequences in at least 19 other bacterial strains (Ruvkun, G. B. and F. M. Ausubel (1980) Proc. Nat. Acad. Sci. U.S.A. 77:191-195). Although this conservation of structure is generally true, significant differences between nitrogenases from different organisms also exist as can be shown by variable stability following purification and by the fact that active hybrid complexes do not form in all cases (Eady, R. R. and B. E. Smith (1979) supra).
A DNA fragment carrying the Klebsiella pneumoniae nifK, nifD and nifH genes has been isolated from the nif-strain UNF841(Tn5:nifK) and cloned into the Escherichia coli plasmid pBR325. The nucleotide sequences of the nifH gene and of 622 nucleotides of the nifD gene were determined (Scott, K. F. et al. (1981) supra). In addition, the DNA sequence of the nifH gene from Anabaena 7120 has been determined (Mevarech, M., Rice, D. and R. Haselkorn (1980) Proc. Nat. Acad. Sci. U.S.A. 77:6476-6480). A comparison of the two nucleotide sequences demonstrates two interesting features: (1) There is very little homology (31%) between the two sequences although a few stretches (up to 25 bp) are conserved, accounting for the observed interspecies homology of the nifH genes (Ruvkun, G. B. and F. M. Ausubel (1980) supra). (2) In general, the promoter regions show very little sequence homology with the exception of a short region likely to be involved in common functions, e.g., RNA polymerase recognition.
In contrast, a comparison of the amino acid sequences of the dinitrogenase reductase and of the first 207 amino acids of the .alpha.-subunit of dinitrogenase of the two species and of another species show a much greater conservatism. The three species used in this comparison are Klebsiella pneumoniae (Kp); Anabaena 7120 (Ab); and Clostridium pasteurianum (Cp) (Tanaka, M. et al. (1977) J. Biol. Chem. 252:7093-7100). The Kp and Cp proteins share 67% amino acid sequence homology, Kp and Ab proteins share 71% homology, and the Cp and Ab proteins share 63%. This amino acid sequence homology is not spread evenly throughout the protein. Some regions are virtually identical--90% to 95% homology), while other regions are only weakly conserved (30-35% homology). The structural conservation appears to be centered around the five cysteine residues common to all three Fe proteins. These cysteine residues are believed to be ligands to the active center.
Comparison of the N-terminal amino acid sequence of the .alpha.-subunit of dinitrogenase from Cp and Kp shows very little sequence homology in this region. This is in contrast to the very high conservation of amino acid sequence seen in the amino terminal region of the Fe protein. What little homology exists between Cp and Kp .alpha.-subunits is confined to regions around cysteine residues, as in the Fe proteins. These homologous regions are thought to be involved in the catalytic functions of the nitrogenase enzyme complex. Therefore, this structural conservatism is thought not to be the result of recent evolution and dispersal of the nif genes (Postgate, J. R. (1974) Sym. Soc. Gen. Microbiol. 24:263-292) but, rather, is postulated to be related to a conservation of function.
The isolation of some of the genes involved in symbiotic nitrogen fixation in R. trifolii has involved a combination of transposon-induced mutagenesis, rapid screening for in planta specific symbiotic mutants, molecular cloning of the mutant symbiotic gene sequences and subsequent isolation of the corresponding wild type DNA fragment from an R. trifolii gene bank. The presence of a wild type symbiotic gene on the cloned DNA fragment was then confirmed by introducing it into its allelic (symbiotically defective) R. trifolii mutant strain and assaying for the restoration of the symbiotic phenotype (Scott, K. F. et al. (1982) J. Mol. Appl. Genetics 1:315-326). The transposon Tn5 was introduced into R. trifolii by conjugation with E. coli strain 1830. Symbiotically defective mutants were recovered by selecting for kanamycin resistant strains. These transposon induced mutants were screened once on plants to determine which of the colonies carrying the Tn5 insertions were symbiotically defective. The phenotype of recovered mutants varied from the complete loss of ability to nodulate (nod-) to the production of nodules with varying morphology but inability to fix nitrogen (fix5-4). Two nod.sup.+ fix.sup.- mutants were specifically reported.
DNA isolated from various Tn5-induced symbiotic mutants was digested with EcoRI cleaved pBR322 plasmid DNA. Tn5 contains no EcoRI restriction sites. These recombinant plasmids were transformed into E. coli RRI and the cells carrying Tn5 and flanking R. trifolii sequences were selected by kanamycin resistance encoded on the transposon. The Tn5-containing recombinant plasmid DNA's were isolated, labelled in vitro with .sup.32 P, and used as probes to identify and isolate corresponding wild type sequences. These cloned fragments were also used to examine the extent of symbiotic gene sequence homology in related species. Homologous sequences were found in some of the fast-growing strains tested, but not in the slow-growing B. japonicum. To isolate wild type DNA sequences corresponding to the mutant sequences cloned from different kanamycin-resistant symbiotically defective mutants, several clone banks of DNA fragments from R. trifolii were generated.
To demonstrate the validity of this approach in the isolation of wild type nitrogen fixation symbiotic gene sequences, two presumptive wild types were analyzed for their ability to "correct" the original Tn5-induced lesion in the R. trifolii genome. Two EcoRI fragments of 6kb and 8kb, respectively, carrying the presumptive wild type sequences were first subcloned into the broad host range conjugative plasmid RP4. These recombinant RP4 plasmids were then conjugated from E. coli RRI into the corresponding R. trifolii mutant (i.e., the mutant used to isolate the presumptive wild type) and cells carrying the cloned symbiotic gene were assayed for their ability to carry out a normal nitrogen-fixing symbiosis on clover plants. Assuming that the cloned DNA fragment carried wild type symbiotic gene sequences, it would be expected that the original mutants would be "corrected" by one of two mechanisms. If the cloned DNA carried the complete symbiotic gene, then correction would occur by complementation and every R. trifolii cell carrying the recombinant RP4 would be capable of an effective symbiotic response. However, if the cloned DNA fragment carried only a portion of the symbiotic gene, then correction of the defect could only occur if the mutant sequences in the genome were replaced by those carried on the RP4 plasmid via homologous recombination. In this case, only a few cells, carrying the recombinant RP4, would be capable of an effective symbiotic response. Both types of responses were observed with different cloned strains. The 6 kb isolate appeared to contain a complete symbiotic gene; however, the larger, 8 kb isolate did not contain all of the information necessary to overcome the symbiotic defect of the Tn5-induced mutant clone to which it hybridized.
In general, identification of a bacterial isolate as a rhizobial species has only been previously achieved by demonstration of nodule formation and re-isolation of the same bacterium from the nodules. Furthermore, due to the specificity of the bacterium-host plant interaction, a number of different legume species must be tested. There are a number of characteristics, however, that clearly indicate that a bacterial isolate is a non-rhizobial species (Vincent, J. M., Nutman et al. (1979) in "Identification methods for Microbiologists", F. A. Skinner and D. W. Lovelock, eds. Academic Press, London). Rhizobia are short to medium gram-negative rods so contra-indications include gram positiveness, endospores, large rods or cocci and chain formation. A rapid growth rate in one or two days and the production of color are also indications that the isolate is not a rhizobial species.
Rhizobial isolates can easily be obtained from freshly-collected turgid nodules of a healthy plant. Rhizobial species can also be isolated from soil but in this case, other bacteria are likely to swamp out the rhizobial species on growth media. If rhizobial species are to be isolated from soil, then it is generally desirable to use a legume species as a "trap" host. Again, due to the specificity of the bacterium-host interaction, a number of different legume species must be used or many rhizobial isolates will be lost. It has been stated by an authority on the subject of symbiotic bacteria-plant relationships (J. M. Vincent (1982) in "Nitrogen fixation in legumes" Academic Press, New York, pp. 5-11) that "except under special circumstances (such as when working with an identifiable ("labelled") strain the ability to nodulate a legume remains the final arbiter as to a culture's allocation to the genus Rhizobium and, in some cases, species." Cross-inoculation patterns have been observed among rhizobial species and proposed as a basis for taxonomic groupings. See Lieberman, M. T. et al. (1985), "Numerical Taxonomic Analysis of Cross-inoculation Patterns of Legumes and Rhizobium," Plant Soil 84:225,244.
A number of methods which are suitable for testing nodulation of large and small seeded species have been described (Vincent, J. M. (1970) "A manual for the practical study of root-nodule bacteria" IBP Handbook No. 15, Blackwell Scientific Publications, Oxford). However, if these tests are not meticulously done, the results are unreliable. For example, a slow-growing and a fast-growing species may occur as a mixture and it is quite easy for the cells of the slow-growing form to remain unobserved within a large, gummy colony. In any event, the tests disclosed in the prior art are time-consuming and tedious.
Rhizobium trifolii strains specifically infect and nodulate clover plants. These strains contain a number of large plasmids ranging in size from about 180 kilobases (kb) to greater than 500 kb. The plasmids can be separated and it has been shown that nitrogen fixation (nif) genes are located on a particular plasmid. This plasmid is therefore referred to as the Sym (symbiotic) plasmid (Hooykaas, P. J. J. et al. (1981) Nature 291:351). It is apparent that no quick and reliable method exists which allows identification of a specific symbiotic plasmid occurring in a rhizobial species with a limited host range.
In many cloning projects, only one of the two DNA strands is required initially. Many techniques have been used including poly(UG)-CsCl gradients (Szybalski, W. et al. (1971) Methods Enzymol., Grossman, L., and Moldave, K., eds. Vol. 21D Academic Press, New York pp. 383-413), polyacrylamide gels (Maxam, A. and W. Gilbert (1977) Proc. Nat. Acad. Sci. U.S.A. 74:560-564), and exonuclease treatment (Smith, A. J. H. (1979) Nucl. Acids. Res. 6:831-848). An alternative biological approach has been developed using the bacteriophage M13. The replicative form of this phage DNA is a circular double stranded molecule; it can be isolated from infected cells and used to clone DNA fragments after which it can be reintroduced into Escherichia coli cells by transfection. M13 phage particles each containing a circular single stranded DNA molecule are extruded from infected cells. Large amounts of single stranded DNA containing a cloned fragment (5-10 .mu.g phage DNA/ml bacterial culture can be easily and quickly recovered (Messing, J. et al. (1977) Proc. Nat. Acad. Sci. U.S.A. 74:3642-3646). The cloning of DNA fragments into the replicative form of M13 has been facilitated by a series of improvements which led initially to the M13mp7 cloning vehicle (Messing, J., Crea, R. and P. H. Seeburg (1981) Nucleic Acids Res. 9:309-321). A fragment of the E. coli lac operon (the promoter and N-terminus of the .beta.-galactosidase gene) was inserted into the M13 genome. A small segment of DNA containing a number of restriction cleavage sites was synthesized and inserted into the structural region of the .beta.-galactosidase fragment. The M13mp7 DNA remains infective and the modified lac gene can still encode the synthesis of a functional .beta.-galactosidase .alpha.-peptide.
The synthesized DNA fragment contains two sites each for the EcoRI, BamHI, SalI, AccI and HincII restriction enzymes arranged symmetrically around a centrally located PstI site. Therefore, by chance, either strand of a cloned restriction fragment can become part of the viral(+) strand depending on the orientation of the cloned fragment relative to the M13 genome after ligation. Insertion of a DNA fragment into one of these restriction sites can be readily monitored because the .alpha.-peptide will be non-functional, so that there will be no .beta.-galactosidase activity.
Following M13mp7, two new single stranded DNA bacteriophage vectors, M13mp8 and M13mp9, have been constructed (Messing, J. and J. Vieria (1982) Gene 19:269-276). The nucleotide sequence of M13mp7 has been modified to contain only one each of the restriction sites (instead of two) and single restriction sites for HindIII, SmaI and XmaI have been added. Thus the restriction sites are EcoRI-SmaI-XmaI-BamHI-SalI-AccI-HincII-PstI-HindIII. These restriction sites have opposite orientations in M13mp8 and M13mp9. DNA fragments whose ends correspond to two of these restriction sites can be "force cloned" to one or the other of these two M13 cloning vehicles which have also been "cut" by the same pair of restriction enzymes. Thus a DNA fragment can be directly oriented by forced cloning. This procedure guarantees that each strand of the cloned fragment will become the (+) strand in one or the other of the clones and will be extruded as single stranded DNA in phage particles.
Restriction endonuclease cleavage fragments with non-complementing ends are seldom joined in a ligation. DNA cleaved by two different restriction endonucleases therefore cannot be circularized or joined to another fragment produced by the same "two different restriction endonucleases" in both orientations. The result is that a recombinant molecule is formed during the ligation reaction with a defined order of the two fragments. Since the orientation of a cloned DNA fragment in the replicative form of M13 vectors determines which of the two DNA strands is going to be the viral strand, the use of M13mp8 or M13mp9 allows the direct preparation of one of the two DNA strands by cloning.
Co-inventors hereof reported the sequence of the R. trifolii nifH and nifD genes, as well as the 5' region of the nifH gene in Scott, K. F. et al. (1983), "Biological Nitrogen Fixation: Primary Structure of the Rhizobium trifolii Iron Protein Gene," DNA 2:149-155. The possible existence of a repeated sequence from this region was suggested in this article. This article was published less than one year prior to the priority filing dates hereof.